There are a number of microorganisms that utilize single carbon substrates as their sole energy source. Such microorganisms are referred to herein as “C1 metabolizers”. These organisms are characterized by the ability to use carbon substrates lacking carbon to carbon bonds as a sole source of energy and biomass. All C1 metabolizing microorganisms are generally classified as methylotrophs. Methylotrophs may be defined as any organism capable of oxidizing organic compounds that do not contain carbon-carbon bonds. However, facultative methylotrophs, obligate methylotrophs, and obligate methanotrophs are all various subsets of methylotrophs. Specifically:                Facultative methylotrophs have the ability to oxidize organic compounds which do not contain carbon-carbon bonds, but may also use other carbon substrates such as sugars and complex carbohydrates for energy and biomass;        Obligate methylotrophs are those organisms which are limited to the use of organic compounds that do not contain carbon-carbon bonds for the generation of energy; and        Obligate methanotrophs are those obligate methylotrophs that have the distinct ability to oxidize methane.        
The ability of obligate methanotrophic bacteria to use methane as their sole source of carbon and energy under ambient conditions, in conjunction with the abundance of methane, makes the biotransformation of methane a potentially unique and valuable process. As such, several have attempted to harness the unique natural abilities of these organisms for commercial applications. For example, the commercial applications for the biotransformation of methane have historically fallen broadly into three categories: 1.) production of single cell protein; 2.) epoxidation of alkenes for production of chemicals; and 3.) biodegradation of chlorinated pollutants. Of these, only epoxidation of alkenes has experienced some commercial success; however, the success has been limited due to low product yields, toxicity of products, and the large amount of cell mass required to generate products. Large-scale protein production from methane, termed single cell protein (or “SCP”), has been technically feasible and commercialized at large scale (Villadsen, J., Recent Trends Chem. React Eng., [Proc. Int. Chem. React. Eng. Conf.], 2nd ed.; Kulkarni, B. D., Mashelkar, R. A., and Sharma, M. M., Eds.; Wiley East: New Delhi, India (1987); Vol 2, pp 320-33). However, SCP has not been economically successful thus far due to the relatively high cost of producing microbial protein, as compared to agriculturally derived protein (i.e., soy protein). This makes SCP a relatively low value product whose economic production cannot tolerate heavy bioprocessing costs. Thus, the yield of the methanotrophic strain used for producing SCP may be critical to the overall economic viability of the process. Microbial biomass produced by methanotrophic bacteria is typically very high in protein content (˜70-80% by weight), which can restrict the direct use of this protein to certain types of animal feed.
In addition to the synthesis of SCP, methanotrophic cells can further build the oxidation products of methane (i.e., methanol and formaldehyde) into complex molecules such as carbohydrates and lipids. For example, under certain conditions methanotrophs are known to produce exopolysaccharides (U.S. Pat. No. 6,537,786; U.S. Pat. No. 6,689,601; Ivanova et al., Mikrobiologiya, 57(4):600-5 (1988); Kilbane, John J., II, Gas, Oil, Coal, Environ. Biotechnol. 3, [Pap. IGT's Int. Symp.], 3rd ed., Meeting Date 1990; Akin, C. and J. Smith, Eds; IGT: Chicago, Ill. (1991); pp 207-26). Similarly, methanotrophs are known to accumulate both isoprenoid compounds and carotenoid pigments of various carbon lengths (U.S. Pat. No. 6,660,507; U.S. Pat. No. 6,689,601; Urakami et. al., J. Gen. Appl. Microbiol., 32(4):317-41 (1986)).
Most recently, the natural abilities of methanotrophic organisms have been extended by the advances of genetic engineering. Odom et al. have investigated Methylomonas sp. 16a as a microbial platform of choice for production of a variety of materials beyond single cell protein, including carbohydrates, pigments, terpenoid compounds and aromatic compounds (U.S. Pat. No. 6,689,601 and U.S. Ser. No. 09/941,947, herein incorporated entirely by reference). This particular pink-pigmented methanotrophic bacterial strain is capable of efficiently using either methanol or methane as a carbon substrate, is metabolically versatile in that it contains multiple pathways for the incorporation of carbon from formaldehyde into 3-carbon units, and is capable of genetic exchange with donor species such as Escherichia coli via bacterial conjugation. Thus, Methylomonas sp. 16a can be engineered to produce new classes of products other than those naturally produced from methane. Further advancement in the metabolic engineering of this particular host organism for production of various commercial products on an economic scale, however, requires some optimization of the host organism. Specifically, it would be desirable to knockout the native carotenoid pathway of the organism leading to the production of pink-pigmented C30 carotenoids, increasing the available carbon flux directed toward the products of interest. These modified host organisms should preferably lack antibiotic markers, since the presence of antibiotic resistance genes in the modified host organism could be undesirable in many food and feed applications. The problem to be solved, therefore, is to develop an optimized non-pigmented Methylomonas sp. 16a bacterial host organism lacking antibiotic markers for production of various commercial products on an economic scale.
The present problem has been solved through the development of a suite of optimized non-pigmented Methylomonas sp. 16a bacterial host organisms, each lacking antibiotic markers. These bacterial hosts were created by investigation of allelic exchange mutations within the native crt gene cluster (comprising the crtN1, ald, and crtN2 genes) and the crtN3 gene of Methylomonas sp. 16a, each of which is associated in the biosynthesis of native C30 carotenoids in the organism. An efficient means of generating defined mutants by homologous recombination permitted transformants that have undergone allelic exchange to be selected based on a positive selection strategy. This methodology also enabled production of “markerless” transformants and permitted multiple rounds of mutation to be performed.